ABSTRACT Interferon regulatory factor 4 (IRF4) is an attractive therapeutic target in multiple myeloma (MM). We here report that expression of IRF4 mRNA inversely correlates with microRNA

(miR)-125b in MM patients. Moreover, we provide evidence that miR-125b is downregulated in TC2/3 molecular MM subgroups and in established cell lines. Importantly, constitutive expression of

miR-125b-5p by lentiviral vectors or transfection with synthetic mimics impaired growth and survival of MM cells and overcame the protective role of bone marrow stromal cells _in vitro_.

Apoptotic and autophagy-associated cell death were triggered in MM cells on miR-125b-5p ectopic expression. Importantly, we found that the anti-MM activity of miR-125b-5p was mediated via

significant anti-tumor activity and prolonged survival. Taken together, our findings provide evidence that miR-125b, differently from other hematologic malignancies, has tumor-suppressor

activity in MM. Furthermore, our data provide proof-of-concept that synthetic miR-125b-5p mimics are promising anti-MM agents to be validated in early clinical trials. SIMILAR CONTENT BEING

VIEWED BY OTHERS THERAPEUTIC TARGETING MIR130B COUNTERACTS DIFFUSE LARGE B-CELL LYMPHOMA PROGRESSION VIA OX40/OX40L-MEDIATED INTERACTION WITH TH17 CELLS Article Open access 18 March 2022

ANTI-BACTERIAL AND ANTI-VIRAL NANCHANGMYCIN DISPLAYS ANTI-MYELOMA ACTIVITY BY TARGETING OTUB1 AND C-MAF Article Open access 30 September 2020 AN MTORC1 TO HRI SIGNALING AXIS PROMOTES

CYTOTOXICITY OF PROTEASOME INHIBITORS IN MULTIPLE MYELOMA Article Open access 18 November 2022 INTRODUCTION Multiple myeloma (MM) is a genetically complex malignancy from the outset, with

progressive acquisition of genetic lesions mediating drug resistance and high disease burden.1 Despite recent progress in the understanding MM pathobiology and the availability of innovative

drugs which have improved clinical outcome, the disease eventually progresses to a drug-resistant lethal stage (plasma cell leukemia)2, 3, 4 and novel therapeutic strategies are therefore

eagerly awaited. Indeed, one of the major challenges in treating MM is its genomic and phenotypic heterogeneity.5 Hence, an optimal therapy would target an essential regulatory pathway

shared by all disease subsets.6 Interferon regulatory factor 4 (IRF4) is a lymphocyte-specific transcription factor.7 Interference with IRF4 expression is lethal for MM cells, irrespective

of their genetics, making IRF4 an ‘Achilles’ heel’ that may be exploited therapeutically.8 Specifically, IRF4 is oncogenic and overexpressed when translocated to actively transcribed genomic

regions in some MM patients, but it also has a survival effect in MM cells in the absence of translocations or overexpression.7, 8 A relevant IRF4 target gene is c-Myc,7, 8 which has a

prominent role in the pathogenesis of MM.7, 8 Another downstream IRF4 effector is B-lymphocyte-induced maturation protein-1 (BLIMP-1):9 indeed, knockdown of BLIMP-1 causes apoptosis in MM

cells. These findings suggest that IRF4 may regulate MM cell survival through modulation of BLIMP-1.9 Moreover, it has been recently demonstrated that caspase-10 (casp-10) and cFlip genes

are transactivated by IRF4: importantly, the evidence that all MM cell lines require casp-10 and cFLIP for survival led to the hypothesis that loss of the proteolytic activity of the

casp-10/cFlip heterodimer mediates MM cell death induced by IRF4 knockdown.10 All these data indicate IRF4 as an attractive therapeutic target in MM. However, efficient _in vivo_ strategies

aimed at blocking IRF4 pathway are still lacking. MicroRNAs (miRNAs) are small non-coding RNAs of 19–25 nucleotides, which regulate gene expression by degrading or inhibiting translation of

target mRNAs, primarily via base pairing to partially or fully complementary sites in the 3′ untranslated region (UTR).11 Targeting deregulated miRNAs in cancer cells is emerging as a novel

promising therapeutic approach,12, 13, 14 including in MM.15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 In this scenario, replacement of tumor-suppressor

miRNAs by synthetic oligonucleotides (miRNA mimics) offers a new therapeutic opportunity to restore a loss-of-function in cancer, that has been an unmet need for drug developers.35 Here, we

show that IRF4 expression is regulated by microRNA-125b-5p (miR-125b-5p) in patient-derived MM cells and MM cell lines. In most of these cells, enforced expression of miR-125b-5p affects

growth and survival, acting via IRF4 downregulation and impairment of its downstream signaling. Overall, our findings demonstrate that miR-125b is a tumor suppressor in MM, and provide the

rationale for development of miR-125b-5p mimics as novel therapeutics. MATERIALS AND METHODS MM PATIENT CELLS AND CELL LINES Following the Magna Graecia University IRB study approval,

primary MM cells were isolated from bone marrow (BM) aspirates, as described,19 from 24 newly diagnosed MM patients who had provided the informed consent. For transfection purposes and

proliferation/survival assays, peripheral blood mononuclear cells (PBMCs) from healthy donors have been used as controls. MM cell lines were cultured as described.19 HS-5 human stromal cell

line (purchased from ATCC, Manassas, VA, USA, CRL-11882) was cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (see

Supplementary Methods for detailed information). VIRUS GENERATION AND INFECTION OF CELLS Cells stably expressing green fluorescent protein transgene were obtained as described.21 To generate

cells stably expressing luciferase transgene, NCI-H929 cells were transduced with pLenti-III-PGK-Luc (ABM Inc., Richmond, BC, Canada) vector. MM cells stably expressing miR-125b-1 and

miR-125b-2 genes were transduced with Lenti-miR-125b-1 and Lenti-miR-125b-2 miRNA precursor constructs (System Biosciences, CA, USA); lentiviral particles were produced and transduced as

previously described.19 RNA EXTRACTION AND QRT-PCR. RNA samples of healthy donors BM-derived plasma cells were purchased (AllCells LLC, Alameda, CA, USA). Total RNA extraction from MM cells

and quantitative real-time PCR were performed as previously described (see Supplementary Methods for detailed information).19 _IN VITRO_ TRANSFECTION OF MM CELLS Synthetic miRNA mimics were

purchased from Ambion (Applied Biosystems, Carlsbad, CA, USA), while synthetic miRNA inhibitors were purchased from Exiqon (Vedbaek, Rudersdal, Denmark). Silencer Select siRNAs were

purchased from Ambion (Applied Biosystems). All the oligos were used at 100 nmol/l final concentration. A total of 2,5 × 105 cells were transfected using Neon Transfection System

(Invitrogen, Carlsbad, CA, USA) (2 pulse at 1.050 V, 30 ms) and the transfection efficiency, evaluated by flow-cytometric analysis relative to a FAM dye-labeled miRNA inhibitor negative

control, reached 85 to 90%. The same conditions were applied for transfection of MM cells with 5 μg of expression vectors carrying the open reading frames (ORFs) of BLIMP-1 (EX-Z5827-M11),

IRF4 (EX-M0891-M11) or empty vector (EX-EGFP-M11) (GeneCopeia, Rockville, MD, USA). SURVIVAL ASSAY Cell viabilty was evaluated by Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular

Technologies, Mashikimachi, Japan) and 7-Aminoactinomycin (7-AAD) flow cytometry assay (BD Biosciences, San Jose, CA, USA), according to manufacturer’s instructions. DETECTION OF APOPTOSIS

Apoptosis was investigated by three different assays: Annexin V/7-AAD flow cytometry assay, terminal-deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay and western blot

analysis of caspases expression and cleavage. To perform TUNEL assay, transfected cells were fixed using 4% paraformaldehyde and then permeabilized using 0.25% Triton X-100, according to

manufacturer’s instructions. Cells were stained with a TUNEL assay (Click-iT TUNEL Alexa Fluor 594 Imaging Assay, Invitrogen, 10246) to identify those with fragmented DNA. Nuclei were

counterstained with Hoechst 33342 (Life Technologies, CA, USA). Image acquisition was done using EVOS FLOID (Life Technologies) equipped with a 20 × Nikon objective. WESTERN BLOT ANALYSIS

Whole cell protein extracts were prepared from MM cell lines and from PBMCs in NP40 CellLysis Buffer (Life Technologies) containing a cocktail of protease inhibitors (Sigma, Steinheim,

Germany). Cell lysates were loaded and polyacrylamide gel electrophoresis separated. Proteins were transferred by Trans-Blot Turbo Transfer Starter System (Bio-Rad, Hercules, CA, USA) for 7 

min. After protein transfer, the membranes were blotted with the primary antibodies (see Supplementary methods for detailed information). ANIMALS AND _IN VIVO_ MODEL OF HUMAN MM Male CB-17

Severe Combined Immunodeficient (SCID) mice (6–8 weeks old; Harlan Laboratories, Inc., Indianapolis, IN, USA) were housed and monitored in our Animal Research Facility. Experimental

procedures and protocols had been approved by the Magna Graecia University IRB and conducted according to protocols approved by the National Directorate of Veterinary Services (Italy, Rome).

Mice were s.c. inoculated with 5 × 106 NCI-H929 cells and treatment started when palpable tumors became detectable. Sample size (that is, number of animals to be inoculated with MM cells)

was chosen accordingly to our experience.16, 17, 18, 19, 20 Tumor sizes were measured as described,19 and the investigator was blinded to group allocation. Tumor size of luciferase

gene-marked NCI-H929 xenografts was also measured by IVIS Lumina II (PerkinElmer, Waltham, MA, USA). Oligos were NLE-formulated within MaxSuppressor _In Vivo_ LANCEr II (BIIo Scientific,

Austin, TX, USA) to achieve an efficient delivery, as reported.16, 27 STATISTICAL ANALYSIS Each experiment was performed at least three times and values are reported as mean±s.d. Comparisons

between groups were made with student’s _t_-test, while statistical significance of differences among multiple groups was determined by GraphPad software (www.graphpad.com). Graphs were

obtained using Graphpad Prism version 6.0 (GraphPad Software, La Jolla, CA, USA). _P_-value <0.05 was accepted as statistically significant. RESULTS INVERSE CORRELATION BETWEEN IRF4 MRNA

AND MIR-125B IN MM PATIENTS To identify IRF4-targeting miRNAs, we interrogated microRNA Data Integration Portal (mirDIP), applying the _high precision_ quality filter.36 As shown in Table 1,

this analysis disclosed 12 mature miRNAs including miR-125a-5p, miR-125b-5p, miR-128, miR-27a-3p, miR-27b-3p, miR-30a-5p, miR-30b-5p, miR-30c-2, miR-30d-5p, miR-30e-5p, miR-4319 and

miR-513a-5p. We next attempted to correlate the expression of these miRNAs and IRF4 mRNA in our dataset (55 MM and 21 plasma cell leukemia patients)(GSE39925). This integrated approach

revealed a significant inverse correlation between IRF4 mRNA and five precursor-miRNAs (pre-miR-125b-1, pre-miR-125b-2, pre-miR-30b, pre-miR-30c-2 and pre-miR-30d) among all MM and plasma

cell leukemia patients examined (Figures 1a and b and Supplementary Fig. S1A). Importantly, when a second dataset (GSE47552) was interrogated, the inverse correlation with IRF4 mRNA was

confirmed only for pre-miR-125b-1 (Supplementary Fig. S1B), strengthening a possible role of miR-125b-5p as IRF4 negative regulator in MM patients. By qRT-PCR, we then evaluated the

expression of miR-125b-5p in 24 CD138+ primary patient MM (ppMM) cells, 10 MM cell lines and 3 samples of CD138+ BM-derived plasma cells from healthy donors (HD-PCs). A significant

downregulation of miR-125b-5p was found in MM cell lines (Figure 1c, with single values plotted in Supplementary Fig. S2), while the downregulatory trend observed in ppMM cells reached

statistical significance within the TC2 and TC3 subgroups only (Figures 1c and d). ENFORCED EXPRESSION OF MIR-125B IMPAIRS GROWTH AND SURVIVAL OF MM CELLS To evaluate the effects induced by

miR-125b, we transduced 3 MM cell lines (NCI-H929, SK-MM-1, RPMI-8226) with lentiviral vectors carrying either miR-125b-1 or miR-125b-2 (Lenti-miR-125b-1 or Lenti-miR-125b-2) genes. The

effects on cell proliferation were assessed by CCK-8 assay at 2, 3 and 4 days after selection by puromycin. As shown in Figures 2a and b, constitutive expression of either Lenti-miR-125b-1

or Lenti-miR-125b-2 resulted in a strong inhibition of cell growth. Next, we transfected MM cell lines with either synthetic miR-125b-5p mimics or inhibitors. We found that ectopic

expression of miR-125b-5p inhibitors did not affect the proliferation of MM cells (Figures 2c and d); conversely, transfection of miR-125b-5p mimics strongly impaired growth and survival of

most MM cell lines (9 out of 10) (Figures 2e and f). Importantly, miR-125b-5p mimics reduced the viability of ppMM cells from 3 individuals (Figure 2g), but not of PBMCs from 6 healthy

donors (Figure 2h). Taken together, these results indicate that enforced expression of miR-125b-5p inhibits growth and survival of MM cells, consistent with a tumor-suppressor function of

this miRNA. Notably, baseline expression of miR-125b-5p did not correlate with the sensitivity/response to synthetic mimics or inhibitors, suggesting that miR-125b-5p expression is not

predictive of _in vitro_ anti-MM activity. Furthermore, miR-125b-5p mimics were lethal to MM cells irrespective of their genetics. MIR-125B-5P MIMICS INHIBIT PROLIFERATION OF MM CELLS VIA

TARGETING IRF4 To investigate whether IRF4 expression could be affected by ectopic miR-125b-5p, both qRT-PCR and western blot analysis were performed in 3 MM cell lines transfected with

miR-125b-5p mimics or scrambled controls (miR-NC). Specifically, the IRF4 translocated SK-MM-1, along with NCI-H929 and RPMI-8226 cells (not IRF4 translocated), were selected for this

analysis. As shown in Figures 3a and b, transfection of miR-125b-5p downregulated IRF4 at both mRNA and protein levels in all MM cell lines. Notably, the only MM cell line resistant to

miR-125b-5p overexpression lacked detectable IRF4 (i.e. RPMI-8226/Dox40 cells, which were generated from the parental RPMI-8226 by continuous exposure to increasing amounts of doxorubicin to

culture medium, is characterized by loss of miR-125b-5p relevant targets) (Supplementary Fig. S3A). Consistently, we found that viability of RPMI-8226/Dox40 cells was not affected by IRF4

siRNA silencing (Supplementary Fig. S3B); in contrast, siRNA-transfection of both NCI-H929 and SK-MM-1 cells confirmed its role in supporting their survival (Supplementary Fig. S3B). We next

investigated whether ectopic expression of a cDNA containing only the coding region of IRF4 and lacking the miR-125b-5p-targeted 3′ UTR could protect MM cells from miR-125b-5p

anti-proliferative effects. As shown in Figure 3c, transfection of IRF4 construct increased IRF4 protein expression which was not affected by miR-125b-5p mimics. Importantly, IRF4

overexpression rescued SK-MM-1 cells from the growth-inhibitory activity of either IRF4 siRNAs (Supplementary Fig. S3C) or miR-125b-5p mimics (Figure 3d), indicating that miR-125b-5p exerts

its anti-MM activity via targeting IRF4. ENFORCED EXPRESSION OF MIR-125B-5P IMPAIRS IRF4 SIGNALING IN MM CELL LINES We next investigated the effects of miR-125b-5p on the molecular network

underlying IRF4 activity in MM. Interestingly, the IRF4-downstream effector BLIMP-1 has been proven to be a direct target of miR-125b-5p.37 As shown in Figure 4a and Supplementary Fig. S4A,

transfection of miR-125b-5p mimics downregulated BLIMP-1 protein in SK-MM-1 and NCI-H929 cells. Thus, we investigated whether ectopic expression of a cDNA containing only the coding region

of BLIMP-1 and lacking the miR-125b-5p-targeted 3′ UTR could protect MM cells from miR-125b-5p effects. Importantly, co-transfection with BLIMP-1 construct weakened the anti-proliferative

effect of miR-125b-5p mimics as well of BLIMP-1 siRNAs (Figure 4c and Supplementary Fig. S4B), indicating that also BLIMP-1 mediates the anti-MM activity of miR-125b-5p. Of note,

miR-125b-5p-induced downregulation of BLIMP-1 was not abrogated in SK-MM-1 cells co-transfected with the coding region of IRF4 (Figure 4b), consistent with the notion that BLIMP-1 is a

direct target of miR-125b-5p which circumvents IRF4 activity on BLIMP-1. Other IRF4-downstream effectors, with a prominent role in MM pathogenesis, are c-Myc, casp-10 and cFLIP.8, 10 By

western blot analysis, we found reduced expression of these proteins in SK-MM-1 (Figure 4d) and NCI-H929 (Supplementary Fig. S4E) cells at 24–48 h after transfection with miR-125b-5p.

Expression of both casp-10 and cFlip was also reduced at mRNA levels (Supplementary Fig. S4F) by miR-125b-5p mimics. _In silico_ search for target prediction identified both casp-10 and

cFlip as _bona fide_ direct targets for miR-125b-5p. To validate this interaction in MM cells, SK-MM-1 cells were co-transfected with miR-125b-5p mimics or scrambled oligonucleotides,

together with an expression vector carrying the 3′ UTR of casp-10 or cFlip mRNA cloned downstream of luciferase reporter gene. Of note, we did not find significant changes in 3′ UTR

luciferase activity after miR-125b-5p overexpression, ruling out direct targeting of casp-10 and cFlip mRNAs by miR-125b-5p (Supplementary Fig. S4G). Moreover, miR-125b-5p-induced

downregulation of c-Myc, casp-10 and cFlip was abrogated in SK-MM-1 cells co-transfected with the coding region of IRF4 (Figure 4e), indicating that miR-125b-5p-induced downregulation of

casp-10, c-Myc and cFlip occurs via IRF4 inhibition. Taken together, our findings demonstrate that miR-125b-5p mimics impair IRF4 signaling in MM cells (Figure 4f). We also found that direct

targeting of IRF4 or BLIMP-1 and indirect modulation of c-Myc, casp-10 and cFlip mediates anti-MM activity of this miRNA. MIR-125B-5P MIMICS TRIGGER BOTH APOPTOTIC AND AUTOPHAGY-ASSOCIATED

CELL DEATH Inhibition of IRF4, as well of BLIMP-1 or c-Myc, has been mainly related to induction of apoptosis,9, 38 while targeting casp-10 or its interaction with cFlip triggers autophagic

cell death of MM cells.10 On this basis, we next determined whether apoptotic or autophagy-associated cell death occurred in MM cells with enforced expression of miR-125b-5p. Using Annexin

V/7-AAD flow cytometry assay, we found that miR-125b-5p mimics triggered exposure of phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the cell surface of SK-MM-1 (Figure 5a),

NCI-H929 and U266 cells (Supplementary Fig. S5A), followed by cell membrane disruption. Moreover, by TUNEL assay and western blotting, we found that miR-125b-5p induced DNA-fragmentation

(Supplementary Fig. S5B) and cleavage/activation of both initiator casp-8 and effector casp-3 (Figure 5b and Supplementary Fig. S5C) in MM cells. Of note, apoptosis was not detected when

miR-125b-5p-transfected cells were treated with the pan-caspase inhibitor Z-VAD-fmk (Figure 5c). Overall, these results indicate that miR-125b-5p is pro-apoptotic in MM cells. Moreover, by

flow cytometry analysis of Cyto-ID stained cells, we observed an increase of autophagic vacuoles in SK-MM-1, XG-1 and INA-6 cells, at 48 h after transfection with miR-125b-5p (Figure 5d).

Importantly, the increase of autophagic vacuoles occurred to a similar extent in cells transfected with miR-125b-5p compared with cells starved for 48 h. Moreover, western blot analysis

showed decreased p-62/SQSTM1 and increased Beclin-1 and proteolytic active LCIIIB (Figure 5e), further indicating that miR-125b-5p-induced cell death can be associated with autophagy

induction in MM cells. Indeed, exposure of miR-125b-5p-transfected cells to autophagy inhibitor cloroquine resulted in increased cell death (Figure 5f), thus suggesting a protective role of

autophagy in our experimental settings. MIR-125B-5P MIMICS ANTAGONIZE THE BMSCS PROTECTIVE ROLE ON MM CELLS BM _milieu_ strongly supports survival and proliferation of MM cells.3 In this

regard, we found that exogenous interleukin-6 (IL-6) or insulin-like growth-factor-1 (IGF-1) or hepatocyte growth-factor (HGF) significantly reduced miR-125b-5p expression in all MM cell

lines except U266 cells (Figure 6a), which express L-Myc instead of c-Myc. Since c-Myc suppresses miR-125b transcription39, 40 and is upregulated by IL-6, IGF-1 and HGF,40, 41, 42 we

investigated whether these factors downregulate miR-125b in MM cells through c-Myc induction. Specifically, we treated c-Myc-expressing SK-MM-1 and c-Myc-defective U266 cells with the

10058-F4 small molecule inhibitor of Myc–Max heterodimerization38 and with the JQ1 BET-bromodomain inhibitor, which is reported to inhibit c-Myc transcription.43 Importantly, both compounds

triggered a significant miR-125b-5p upregulation in SK-MM-1 cells, but not in U226 cells (Figure 6b), indicating that c-Myc-independent downregulation of miR-125b occurs in this cell line.

Moreover, IL-6 or IGF-1 or HGF did not affect miR-125b-5p expression in SK-MM-1 cells in the presence of c-Myc inhibitors, indicating a c-Myc-mediated downregulation of miR-125b by exogenous

addition of microenvironmental growth factors (Figure 6c). Finally, we demonstrated that the anti-MM activity of miR-125b-5p mimics was not antagonized by exogenous growth

promoting/pro-survival stimuli including IL-6, IGF-1 and HGF, or by adherence of MM cell lines to HS-5 human stromal cells (Figures 6d and e). This could be partly due to downregulation of

IL-6R/CD126 on the surface of MM cells. Indeed, by flow cytometry analysis, we did observe decreased expression of cell surface IL-6R/CD126, also a further validated target of miR-125b-5p44

(Figure 6f), which translated in reduced levels of phosphorylated/active STAT3 (pSTAT3-Y705) in IL-6-dependent INA-6 cells (Figure 6g). _IN VIVO_ DELIVERY OF NLE-FORMULATED SYNTHETIC

MIR-125B-5P MIMICS EXERTS ANTI-MM ACTIVITY _In vivo_ anti-MM activity of miR-125b-5p was next evaluated in Non-Obese Diabetic (NOD)/SCID mice bearing subcutaneous NCI-H929 xenografts. In our

first model, luciferase gene-marked NCI-H929 xenografts were intra-tumorally treated every other day with 1 mg/kg of oligos for a total of six injections. As shown in Figures 7a–c,

treatment resulted in a significant tumor-growth inhibition and prolonged survival. In a second model, treatments were administered by i.p. injections of formulated oligos (twice weekly, 1 

mg/kg). In this model, we also observed anti-tumor activity of miR-125b-5p, evidenced by growth inhibition and prolonged survival (Figures 7d and e). Importantly, we found increased

miR-125b-5p in tumors retrieved from animals 48 h after treatment, confirming efficient tumor cells uptake of formulated oligos (Figure 7f). Consistent with our _in vitro_ data, IRF4

signaling was correlated with miR-125b-5p activity _in vivo_: downregulation of IRF4 and BLIMP-1 (Figure 7g), along with decreased expression of c-Myc, casp-10 and cFlip proteins (Figure

7h), was detected in tumors retrieved from miR-125b-5p-treated animals. Overall, these data indicate that _in vivo_ anti-MM activity of miR-125b-5p mimics is associated with abrogation of

IRF4 signaling within MM xenografts. DISCUSSION In this study, we investigated the anti-MM activity of the IRF4-targeting miR-125b-5p. IRF4 is in fact an ‘Achilles’ heel’ for MM cells and,

therefore, represents an attractive therapeutic target in this malignancy.7 miRNAs are natural antisense interactors of mRNAs, and the availability of suitable _in vivo_ delivery systems has

recently allowed the development of synthetic miRNA mimics in early clinical trials. By querying mirDIP36 and applying the high precision quality filter, we found 12 mature miRNAs predicted

to target the 3′ UTR of IRF4 mRNA, including miR-125b-5p. Importantly, integrated analysis of miRNAs and mRNAs expression profiles showed that miR-125b, but not the other predicted miRNAs,

inversely correlated with IRF4 mRNA in two different MM datasets, strengthening the relevance of miR-125b as IRF4 negative regulator in MM patients. Furthermore, miR-125b-5p was found

significantly downregulated in patients belonging to TC2 and TC3 molecular subgroups, as well as in MM cell lines. Altogether, these findings prompted us to investigate the role of this

miRNA in MM. miR-125b is one of the most evolutionary conserved miRNAs.45 In humans there are two paralogs (hsa-miR-125b-1 on chromosome 11 and hsa-miR-125b-2 on chromosome 21), coding for

the same mature sequences (miR-125b-5p and miR-125b-3p).45 miR-125b has a crucial role in a variety of cellular processes and diseases.46 It is commonly dysregulated in cancer,46 but its

function diverges in different malignancies, with dependence on the molecular contexts. However, its role in MM is still largely undisclosed.46, 47, 48 We here provide the evidence that

miR-125b acts as a tumor suppressor in MM by targeting IRF4 and BLIMP-1 mRNAs, and importantly we show that NLE-formulated synthetic miR-125b-5p mimics induce anti-tumor activity _in vivo_.

Specifically, we demonstrate that both lentivirus-based constitutive expression of miR-125b-1/-2 genes and transient enforced expression of synthetic miR-125b-5p mimics inhibit the growth

and survival of MM cell lines. Moreover, viability of ppMM cells, but not of PBMCs from healthy donors, was affected by transfection with miR-125b-5p mimics, suggesting a favorable

therapeutic activity. In our study, baseline expression of miR-125b-5p does not correlate with _in vitro_ sensitivity of MM cells to synthetic mimics; consistent with our findings,

increasing evidence demonstrate that cancer cells with normal miRNA expression are indeed susceptible to miRNA treatment.35 Importantly, the anti-MM activity of miR-125b-5p mimics was not

affected by either exogenous growth promoting/pro-survival stimuli including IL-6, IGF-1 or HGF, or by adherence of MM cell lines to BM stromal cells (BMSCs). This is a crucial point taking

into account that the close and dynamic interplay between MM cells and BMSCs leads to activation of signal transduction pathways which promote cell-cycle progression and protection from

apoptosis.3 Furthermore, a c-Myc-mediated downregulation of miR-125b by exposure of MM cells to different growth factors was observed. To date, a variety of oncogenic pathways have been

identified as directly regulated by miR-125b.46, 47 Here, we demonstrate a functional link between this miRNA and the oncogenic IRF4 signaling in MM. IRF4 is a validated target of

miR-125b-5p37, 49 and the relevance of this interaction is well established in both myeloid- and B-cell leukemias, wherein IRF4 acts as a tumor suppressor and miR-125b as a tumor promoter.50

We found that IRF4 expression was downregulated on transfection of MM cells with miR-125b-5p mimics. Among the MM cell lines studied, only miR-125b-5p-resistant RPMI-8226/Dox40 cells did

not express detectable levels of IRF4. Importantly, overexpression of IRF4 was able to rescue SK-MM-1 cells from the growth-inhibitory activity of miR-125b-5p. Altogether, these data

indicate that IRF4 mediates the anti-MM activity of miR-125b-5p mimics _in vitro_, underlying a novel and divergent role of miR-125b-5p/IRF4 axis in MM as compared with other hematological

malignancies. miRNAs function as master regulators of the genome by modulating the expression of tens to hundreds genes, often belonging to the same pathway.35 This mechanism of action

provides advantage to the therapeutic use of miRNAs, since it is consistent with the current vision of cancer as a pathway disease.35 Interestingly, the IRF4-downstream effector BLIMP-1 is

also regulated by miR-125b-5p,37 and we demonstrated that, similar to IRF4 but to a lesser extent, it also mediates the growth-inhibitory activity of miR-125b-5p. Moreover, we analyzed

perturbations occurring in other relevant effectors of IRF4 signaling, including c-Myc, casp-10 and cFlip. c-Myc has a prominent role in the pathogenesis of MM, wherein it represents an

attractive therapeutic target.7, 38, 43 Importantly, ectopic expression of miR-125b-5p significantly decreased c-Myc protein in MM cells which was rescued by co-transfection with the coding

region of IRF4, indicating that IRF4 mediates miR-125b-5p-triggered downregulation of c-Myc. Recently, it has been demonstrated that MM cells further require the IRF4-downstream effectors

casp-10 and cFlip for their survival: indeed, the heterodimeric protease composed of casp-10 and cFLIP proteins has a balancing role among the pro-survival and pro-death effects of

autophagy.10 Consistent with these notions, we found that both casp-10 and cFlip undergo significant downregulation on miR-125b-5p ectopic expression in MM cells, along with increased

autophagic flux. Nonetheless, the role of autophagy as a response to miR-125b-5p overexpression points to further investigation, taking into account that our present data indeed suggest a

protective effect at least in SK-MM-1 cells. Even though _in silico_ search for target prediction indicated both casp-10 and cFlip as _bona fide_ direct targets for miR-125b-5p, we failed to

validate this interaction. However, miR-125b-5p-induced downregulation of casp-10 and cFlip was abrogated by ectopic IRF4 expression, indicating that miR-125b-5p-mediated downregulation of

these two survival factors depends on IRF4 targeting. Finally, we demonstrated the _in vivo_ anti-tumor activity of NLE-formulated miR-125b-5p mimics against human MM xenografts in SCID/NOD

mice. To our knowledge, this is the first evidence of a successful _in vivo_ treatment with miR-125b-5p mimics in a murine xenograft model of human MM, which indeed has important potential

towards clinical applications. We showed that both intra-tumor and i.p. injection of NLE-formulated miR-125b-5p mimics resulted in significant tumor-growth inhibition and prolonged survival.

Moreover, in tumors retrieved from animals treated with miR-125b-5p mimics, a downregulation of its direct targets IRF4 and BLIMP-1, along with a reduction in expression of c-Myc, casp-10

and cFlip proteins, was observed. These findings suggest that the _in vivo_ anti-MM activity of miR-125b-5p mimics is related to the impairment of IRF4 signaling within MM xenografts.

Further _in vivo_ evaluation in preclinical models recapitulating the huBMM, such as the SCID-synth-hu,51 will strengthen the translational value of miR-125b-5p mimics. We think that our

study provides important proof-of-concept findings for the basic strategy of miRNA therapeutics. In fact, we found that the miR-125b-5p/IRF4 axis has a highly disease-specific functional

role in MM, which runs in opposite directions as compared with other hematological malignancies. These findings support the peculiarity of MM BM microenvironment disease scenario, which

opens highly specific therapeutic avenues. An additional important point is that miR-125b-5p strongly inhibits both IRF4 and BLIMP-1, offering a pathway-directed therapeutic tool, which is

still undruggable by alternative approaches. In conclusion, our investigation provides evidence that miR-125b has tumor-suppressor activity in MM and that enforced expression of synthetic

miR-125b-5p mimics induces significant anti-MM activity _in vitro and in vivo_ by IRF4 targeting. Taken together, these results provide the rational framework for development of

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cells. _Leukemia_ 2011; 25: 707–711. Article  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS This work has been supported by funds of Italian Association

for Cancer Research (AIRC), PI: PT. ‘Special Program Molecular Clinical Oncology—5 per mille’ n. 9980, 2010/15. This work has also been supported by a grant from NIH PO1-155258, RO1-124929,

P50-100007, PO1-78378 and VA merit grant IO1-24467. Nicola Amodio was supported by a ‘Fondazione Umberto Veronesi’ post-doctoral fellowship. AUTHOR CONTRIBUTIONS EM, EL, MEGC, MTDM, NA, LB,

AG, UF, MRP, CB and MR performed experiments and analyzed the data; AN provided biological samples; PT and PT conceived the study; EM, PT and PT wrote the manuscript; NCM and KCA provided

critical evaluation of experimental data and manuscript. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Experimental and Clinical Medicine, Magna Graecia University, Salvatore

Venuta University Campus, Catanzaro, Italy E Morelli, E Leone, M E Gallo Cantafio, M T Di Martino, N Amodio, L Biamonte, A Gullà, U Foresta, M R Pitari, C Botta, M Rossi, P Tagliaferri &

 P Tassone * Department of Medical Sciences, University of Milan, Hematology 1, IRCCS Policlinico Foundation, Milan, Italy A Neri * Department of Medical Oncology, Jerome Lipper Multiple

Myeloma Center, Dana-Farber Cancer Institute, Boston, MA, USA N C Munshi & K C Anderson * VA Boston Healthcare System, West Roxbury, Boston, MA, USA N C Munshi * Sbarro Institute for

Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA, USA P Tassone Authors * E Morelli View author

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author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to P Tassone. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no conflict of interest. ADDITIONAL INFORMATION

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ARTICLE CITE THIS ARTICLE Morelli, E., Leone, E., Cantafio, M. _et al._ Selective targeting of IRF4 by synthetic microRNA-125b-5p mimics induces anti-multiple myeloma activity _in vitro_ and

_in vivo_. _Leukemia_ 29, 2173–2183 (2015). https://doi.org/10.1038/leu.2015.124 Download citation * Received: 19 February 2015 * Revised: 27 April 2015 * Accepted: 05 May 2015 * Published:

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